Helichrysum stoechas (L.) Moench Inflorescence Extract for Tomato Disease Management

Helichrysum stoechas is a singular halophyte that has been shown to have anti-inflammatory, antioxidant, and allelopathic properties. In the work presented herein, we have characterized its inflorescences hydromethanolic extract and assessed its antifungal activity for the pre- and postharvest management of tomato crop diseases. Gas chromatography–mass spectrometry characterization of the extract showed that 4-ethenyl-1,3-benzenediol, 2,3-dihydro-benzofuran, quinic acid, 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-1-benzopyran-4-one, 1,6-anhydro-β-D-glucopyranose, catechol, scopoletin, and maltol were the main constituents. The co-occurrence of pyranones, benzenediols, and quinic acids as phytoconstituents of H. stoechas extract resulted in promising in vitro minimum inhibitory concentrations of 500, 375, 500, 187.5, 187.5, and 375 μg·mL−1 against mycelia of Alternaria alternata, Colletotrichum coccodes, Fusarium oxysporum f. sp. lycopersici, Rhizoctonia solani, Sclerotinia sclerotiorum, and Verticillium dahliae, respectively. Further, to assess the potential of H. stoechas inflorescence extract for postharvest tomato crop protection, ex situ tests were conducted against C. coccodes, obtaining high protection at a dose of 750 μg·mL−1. Taking into consideration that the demonstrated activity is among the highest reported to date for plant extracts and comparable to that of the synthetic fungicides tested as positive controls, H. stoechas inflorescence extract may be put forward as a promising biorational and may deserve further testing in field-scale studies.


Introduction
The genus Helichrysum comprises up to 600 species of flowering plants in the Asteraceae family. Helichrysum spp. have been utilized in various folk medicinal systems for addressing fever and inflammation and managing neurologic and digestive disorders [1,2]. Certain healing attributes have been validated by medical science, including its antimicrobial activity [3,4].
In particular, Helichrysum stoechas (L.) Moench, known as Mediterranean strawflower, curry plant, or yellow amaranth, is a fragrant, thermophilous halophyte found in southern Europe. It is a perennial or annual shrub that likes dry, rocky, and sandy areas. It is a hermaphrodite with grayish-green foliage and yields petite spherical yellow inflorescences.

Antifungal Activity
The antifungal susceptibility test results are depicted in Figure 2. In all instances, an increase in H. stoechas extract concentration resulted in a decrease in mycelium radial growth, yielding statistically significant variances. R. solani and S. sclerotiorum,  The antifungal susceptibility test results are depicted in Figure 2. In all instances, an increase in H. stoechas extract concentration resulted in a decrease in mycelium radial growth, yielding statistically significant variances. R. solani and S. sclerotiorum, specifically, exhibited the highest sensitivity to H. stoechas inflorescence hydromethanolic extract, with minimal inhibitory concentrations (MICs) of 187.5 µg·mL −1 . Complete inhibition for C. coccodes and V. dahliae mycelial growth occurred at 375 µg·mL −1 , while a higher dosage of 500 µg·mL −1 was required to inhibit A. alternata and F. oxysporum f. sp. lycopersici growth. Table 3 displays the effective concentrations at 50% and 90% (EC 50 and EC 90 , respectively). Results of mycelial growth inhibition for the three commercial fungicides selected as positive controls are summarized in Table 4. The mancozeb dithiocarbamate fungicide, at a dosage of 150 µg·mL −1 (one-tenth of the recommended amount), exhibited the highest efficacy, inhibiting the growth of all plant pathogens except for A. alternata. At the recommended concentration of 2000 µg·mL −1 , fosetyl-Al organophosphorus fungicide completely inhibited the growth of all fungal species except for A. alternata, F. oxysporum f. sp. lycopersici, and S. sclerotiorum. Conversely, the strobilurin fungicide (azoxystrobin), at a   Results of mycelial growth inhibition for the three commercial fungicides selected as positive controls are summarized in Table 4. The mancozeb dithiocarbamate fungicide, at a dosage of 150 µg·mL −1 (one-tenth of the recommended amount), exhibited the highest efficacy, inhibiting the growth of all plant pathogens except for A. alternata. At the recommended concentration of 2000 µg·mL −1 , fosetyl-Al organophosphorus fungicide completely inhibited the growth of all fungal species except for A. alternata, F. oxysporum f. sp. lycopersici, and S. sclerotiorum. Conversely, the strobilurin fungicide (azoxystrobin), at a recommended dose of 62,500 µg·mL −1 , displayed the lowest efficacy, failing to fully arrest the development of all phytopathogens.  Same letters denote non-significant differences at p < 0.05. Error bars show standard deviations (n = 6). 'C' represents the untreated control (each fungus growing in potato dextrose agar, PDA, medium with only the extraction solvent added).

Ex Situ Postharvest Protection Tests
H. stoechas inflorescence extract was assessed as a protective measure against anthracnose on tomato cv. "Daniela" fruits. Two concentrations were tested: MIC and MIC×2 (375 and 750 µg·mL −1 , respectively). The results are displayed in Figures 3 and 4. In the positive control (C. coccodes inoculated on tomato fruits and treated solely with bidistilled water), fruits showed dark brown, circular, sunken lesions around the inoculation zone, delimited by a circular chlorotic halo and displaying evident soft rot symptoms ten days post-inoculation ( Figure 4b). The average lesion diameter was 42.2 ± 3.7 mm (Table 5). H. stoechas inflorescence extract, at the MIC concentration, inhibited anthracnose on the fruit by 27%, resulting in lesions similar to the positive control ( Figure 4c). However, when the extract was applied at a higher concentration (MIC×2, Figure 4d), anthracnose symptoms were inhibited by >80% compared to the positive control. [24] * In terms of recommended dose, Rd represents 62.5 mg·mL −1 of azoxystrobin (250 mg·mL −1 for Ortiva ® , azoxystrobin 25%), 1.5 mg·mL −1 of mancozeb (2 mg·mL −1 for Vondozeb ® , mancozeb 75%), and 2 mg·mL −1 of fosetyl-Al (2.5 mg·mL −1 for Fosbel ® , fosetyl-Al 80%). The control (PDA only) exhibited a radial growth of the mycelium measuring 75 mm. All mycelial growth values provided are average values (n = 3).

Ex Situ Postharvest Protection Tests
H. stoechas inflorescence extract was assessed as a protective measure against anthracnose on tomato cv. "Daniela" fruits. Two concentrations were tested: MIC and MIC×2 (375 and 750 µg·mL −1 , respectively). The results are displayed in Figures 3 and 4. In the positive control (C. coccodes inoculated on tomato fruits and treated solely with bidistilled water), fruits showed dark brown, circular, sunken lesions around the inoculation zone, delimited by a circular chlorotic halo and displaying evident soft rot symptoms ten days post-inoculation (Figure 4b). The average lesion diameter was 42.2 ± 3.7 mm (Table 5). H. stoechas inflorescence extract, at the MIC concentration, inhibited anthracnose on the fruit by 27%, resulting in lesions similar to the positive control ( Figure  4c). However, when the extract was applied at a higher concentration (MIC×2, Figure 4d), anthracnose symptoms were inhibited by >80% compared to the positive control.

On the Phytochemical Profile
Considering the hydromethanolic extraction mixture's ability to solubilize non-volatile polar compounds that cannot be detected without previous derivatization before carrying out the GC-MS analysis, it is important to exercise caution with the results. In this study, such prior derivatization was not conducted due to drawbacks such as increased procedural preparation time and cost (which would have a negative impact on the economic viability of the crop protection treatment), complex data acquisition, potential impurities, uncertain compound conversion into derivatives, and the use of toxic reagents [25]. On the other hand, the injection of non-volatile compounds may result in eventual damage to the capillary column.
Regarding the reliability of GC-MS identification of extract components, limitations in identifying certain minority compounds were observed, with low quality of resemblance (Qual) values. This suggests that the identification of compounds like 4-pyrimidinol, 6-(methoxymethyl)-2-(1-methylethyl)-; 2-hydroxy-γ-butyrolactone; and 1-acetyl-2-amino-3cyano-7-isopropyl-4-methylazulene may hold some value, but accuracy cannot be guaranteed. The main constituents, except for 4-ethenyl-1,3-benzenediol, had Qual values higher than 87. In the case of this chemical species, identified at a retention time (RT) of 12.7793 min and for which a Qual = 64 was obtained using the NIST11 database, reintegration and indexing using the Wiley database confirmed its presence ( Figure S3 shows a good MS agreement), also supported by infrared vibrational data.
Concerning the chemical profile of the H. stoechas inflorescences extract, important phytochemicals have been pyranones, such as 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-1-benzopyran-4-one, scopoletin, and maltol, and phenolic acid derivatives such as quinic acid. These components do not coincide exactly with those identified by Barroso et al. [7] (quercetin/myricetin and caffeoylquinic acid), but they have an obvious structural analogy. Phytoconstituents not evidenced in previous reports on H. stoechas extracts have been 4-ethenyl-1,3-benzenediol, 2,3-dihydro-benzofuran, 1,6-anhydro-β-D-glucopyranose, and catechol, all with potential antimicrobial properties [35][36][37]. These differences may be tentatively attributed either to variations in the extraction procedure or to individual, genotype-depending differences, location-related intra-varietal differences, and seasonal variations-all of which could significantly influence phytochemical composition and bioactivity. Additionally, the existence of different chemotypes due to minor genetic and epigenetic changes cannot be excluded. In this regard, analyzing the stability and repeatability of the occurrence of individual components would be an essential area of investigation. This subject has not been covered in the study presented herein or in other previous studies on H. stoechas [7][8][9][10][11], highlighting its potential as a line for further research.

Comparison with Other Helichrysum spp. Extracts
H. stoechas has been tested for antifungal activity against human pathogens, exhibiting MIC values of 8 µg·mL −1 against C. albicans and Candida parapsilosis (Ashford) Langeron & Talice for aqueous and ethanol extracts from its aerial parts [4]. Sobhy et al. [9] reported that the H. stoechas apical parts essential oil (0.7% v/w) inhibited C. albicans, but not C. tropicalis and N. glabratus (the ethanolic extract showed no inhibitory activity). However, Roussis et al. [11] discovered that the essential oil derived from the aerial organs of H. stoechas was effective against C. albicans, C. tropicalis, and N. glabratus, with MIC values in the 3.25-6.8 µg·mL −1 range.
In contrast, the antimicrobial activity of related species such as Helichrysum odoratissimum (L.) Sw., Helichrysum patulum (L.) D.Don, Helichrysum italicum (Roth) G. Don, and Helichrysum plicatum DC has been tested against phytopathogenic fungal taxa. Matrose et al. [45] examined the antifungal efficacy of H. odoratissimum and H. patulum ethanol extracts against Botrytis cinerea Pers., observing inhibition percentages of 65% and 51%, respectively, at a dosage of 250,000 µg·mL −1 . The essential oil from the aerial parts of H. italicum was tested against four fungi (namely, A. alternata, Ascochyta rabiei (Pass.) Labr., Aspergillus niger Tiegh., and Fusarium solani var. coeruleum (Lib. ex Sacc.) C.Booth) [46], finding fungistatic MICs in the 6.325 to 50.6 µg·mL −1 range (lower than that reported here). Regarding the antimicrobial properties of the aqueous ethanol extract of H. plicatum, it inhibited the growth of most tested fungi (including A. alternata, Aspergillus flavus Link, Chaetomium sp., Curvularia lunata  [47], which are also lower than the MIC reported for A. alternata in this study. While both hydromethanolic plant extracts and essential oils can be effective at controlling phytopathogens, it is worth noting that hydroalcoholic plant extracts (such as the one discussed in this work) have some advantages over essential oils, including a broader spectrum of activity, less phytotoxicity, easier extraction, and greater stability.

Comparison of Efficacy vs. Other Plant Extracts
A more extensive comparison with the effectiveness of other tested plant extracts against the six fungi studied herein can be found in Table S1 [23,24,33,. However, caution should be exercised in comparing the results due to variations in isolates (or species, Molecules 2023, 28, 5861 9 of 17 in the case of the genus Colletotrichum) across different studies. Further, in studies where multiple plant extracts were tested, those lacking activity were excluded.
Regarding A. alternata, the H. stoechas extract demonstrated the highest activity (MIC = 500 µg·mL -1 ) among the reported literature, except for the aqueous ethanol extract of H. plicatum mentioned above. As for the activity against C. coccodes (MIC = 375 µg·mL −1 ), no direct comparisons were available, but the activity would be among the highest against Colletrotrichum spp., together with those of Zingiber officinale Roscoe rhizomes chloroform extract and Polyalthia longifolia (Sonn.) Thwaites leaves methanol extract, for which inhibition rates of 87.4 and 84% were attained at 400 µg·mL −1 [61].

Conventional Fungicide Comparison
When the antifungal activity of H. stoechas inflorescence extract (Table 3) was compared with that of conventional synthetic fungicides (Table 4), it was found that the extract was generally less effective than mancozeb against all pathogens, except for A. alternata. In the case of this pathogen, H. stoechas extract achieved full inhibition at 500 µg·mL -1 , whereas mancozeb required over 1500 µg·mL -1 . H. stoechas extract led to complete inhibition at concentrations lower than the recommended dose of fosetyl-Al (2000 µg·mL -1 ). Nevertheless, fosetyl-Al was more effective against C. coccodes, with complete inhibition observed at 200 µg·mL -1 vs. 375 µg·mL -1 for H. stoechas extract. Fosetyl-Al, even at the recommended dose, did not fully inhibit A. alternata, F. oxysporum f. sp. lycopersici, and S. sclerotiorum, requiring doses higher than 2000 µg·mL -1 . At the prescribed concentration of 62,500 µg·mL -1 , azoxystrobin failed to completely hinder any of the six fungal pathogens, indicating notably lower efficacy than the plant extract.

Postharvest Protection Tests
Hydromethanolic plant extracts have not been tested for ex situ inhibition of tomato anthracnose caused by C. coccodes or other Colletotrichum spp. Regarding alternative extraction media, R. coriaria aqueous crude extract at 20 µL·mL −1 provided complete protection of tomato fruits against Colletotrichum acutatum J.H.Simmonds after ten days of incubation [100], indicating higher efficacy compared to H. stoechas extract.
Concerning essential oils, cinnamon and clove ones were found to reduce lesion diameter on immature green pepper fruits inoculated with C. gloeosporioides [102]. In another study [103], cinnamon and lemongrass oils were reported to exhibit strong inhibitory activity against C. acutatum on mangoes but caused severe damage to fruit peels, while basil essential oil reduced C. acutatum lesions without harming the fruit.
In terms of innovative application methods, nanoemulsion-based coatings have been proposed as an effective technology for anthracnose control [104]. For example, Oliveira et al. [105] demonstrated that coatings combining chitosan (at 5000 µg·mL −1 ) with Cymbopogon citratus (D.C. ex Nees) Stapf essential oil (0.15-0.6 µL·mL −1 ) exhibited similar or even better efficacy than synthetic fungicides in controlling anthracnose on guava (Psidium guajava L.), mango, and papaya 12 days after inoculation. Similarly, Grande Tovar et al. [106] and Peralta-Ruiz et al. [107] investigated the inhibitory effects of chitosan and Ruta graveolens L. essential oil coatings on guava and papaya fruits infected with Colletotrichum spp., and observed reductions in lesion expansion ranging from 50-67% for a treatment dose of 0.5% to 69-100% for concentrations of 1-1.5%.
These promising findings suggest that H. stoechas extract could be incorporated into chitosan films and coatings in future studies, benefiting from potential synergistic interactions with the biopolymer. Such films and coatings, applied via spray coating or fruit dipping, would be more reproducible and scalable treatment methods for potential industrial application than the one assayed herein. In this regard, regardless of whether the extract is used alone or dispersed in biopolymeric films, the development of formulations based on H. stoechas extracts would require further research and exploration at a more advanced stage.

Plan Material and Extraction Protocol
Aerial parts were collected from H. stoechas plants in June 2022 near the city of Huesca, Spain. The specific location was 42 • 09 15.4 N 0 • 27 50.1 W. The plants were in full bloom at that time. A voucher specimen, verified by Prof. J. Ascaso, was stored in the herbarium of EPS-Universidad de Zaragoza. The inflorescences were separated from stems and leaves. To create representative composite samples, 20 specimens were mixed together. These composite samples were dried in the shade, ground into a fine powder using a mechanical grinder, and then homogenized and sieved through a 1 mm mesh.
The extraction process using ultrasonication was similar to the one described in [31]. The use of a methanol:water (1:1, v/v) extraction medium offers versatility, cost-effectiveness, and efficient extraction of a wide range of phytochemicals. Ultrasound-assisted extraction provides increased extraction efficiency, reduced extraction time, preservation of compound integrity, and energy efficiency. The procedure was as follows: the dried inflorescence sample (19.6 g) was mixed with a methanol:water solution (1:1 v/v; 250 mL). The mixture was heated and stirred for 20 min at 50 • C. It was then sonicated using a model UIP1000 hdT probe-type ultrasonicator from Hielscher Ultrasonics (Teltow, Germany). After sonication, the mixture was centrifuged at 9000 rpm for 10 min. The resulting liquid was filtered through Whatman No. 1 paper and freeze-dried, resulting in a solid residue. The extraction yield was only 0.6%.
For the subsequent GC-MS analysis, the freeze-dried extract was redissolved in methanol (HPLC-grade) to yield a solution with a concentration of 5 mg·mL −1 . The solution was then filtered again.

Characterization Procedures
The infrared vibrational spectrum of the dried inflorescence sample from H. stoechas was measured using an iS50 Fourier-transform infrared (FTIR) spectrometer (Nicolet, Thermo Scientific; Waltham, MA, USA) with an attenuated total reflectance (ATR) system. The range of measurement was 400-4000 cm −1 , with a 1 cm −1 resolution. The resulting spectrum was obtained by combining 64 scans.
The hydroethanolic extract of H. stoechas inflorescence was analyzed using a GC-MS system at the Research Support Services of Universidad de Alicante. The system consisted of a 7890A gas chromatograph coupled to a 5975C quadrupole mass spectrometer (Agilent Technologies; Santa Clara, CA, USA). The following conditions were used for chromatography: injection volume = 1 µL; injector temperature = 280 • C (in splitless mode); and initial oven temperature = 60 • C for 2 min, followed by a ramp of 10 • C/min up to a final temperature of 300 • C for 15 min. Separation of compounds was achieved using an HP-5MS UI column (Agilent Technologies) with a length of 30 m, a diameter of 0.250 mm, and a film thickness of 0.25 µm. The mass spectrometer conditions were as follows: temperature of the electron impact source = 230 • C; temperature of the quadrupole = 150 • C; and ionization energy = 70 eV. Components were identified by comparing their mass spectra and retention time with those of authentic compounds and by utilizing the databases of the National Institute of Standards and Technology (NIST11) and Wiley.

In Vitro Antifungal Activity
The antifungal activity of the H. stoechas aerial part extract was assessed using the poisoned food method [108]. Stock solution aliquots were added to the PDA medium, resulting in final concentrations ranging from 15.62 to 1500 µg·mL −1 . Mycelial plugs coming from one-week-old PDA cultures of A. alternata, C. coccodes, F. oxysporum f. sp. lycopersici, R. solani, S. sclerotiorum, and V. dahliae were transferred to plates containing the amended media. Each treatment and concentration combination utilized three plates, with the experiment repeated twice. The untreated control involved replacing the extract with the solvent used for extraction in the PDA medium (methanol:water, 1:1 v/v). Additional controls including pure PDA medium and PDA with the lowest treatment concentration were included to validate the absence of contamination. Positive controls consisted of commercial fungicides, namely, Ortiva ® , Vondozeb ® , and Fesil ® , and were conducted according to the indications and doses recommended by each manufacturer.
It was decide to segregate the analysis of fungicides from that of the extract evaluation for several reasons: on the one hand, the recommended concentrations of the commercial products are usually significantly different from those used in laboratory standards for antibiotic activity; on the other hand, commercial fungicide products are typically formulated with specific purity levels and often contain additional substances that enhance their effectiveness (and, consequently, their dose-response curve), while plant extracts are complex matrices of several active components, where the adjustment of the final concentrations employed are made on the whole in each specific extract.
In all bioassays, radial mycelium growth was evaluated by measuring the average of two colony diameters that were perpendicular to each other for every repetition. Growth suppression was determined using the following formula after a one-week incubation in complete darkness at a temperature of 25 • C: ((d c − d t )/d c ) × 100, where d c denotes the mean colony diameter in the untreated control, and d t represents the mean diameter of the treated colony. The effective concentrations were estimated by fitting them to a four-parameter logistic equation (dose-response curve). The mycelial growth inhibition results were analyzed in IBM SPSS Statistics v.25 (IBM; Armonk, NY, USA) using analysis of variance (ANOVA), followed by Tukey's test for post hoc comparison of means, as the Shapiro-Wilk and Levene tests confirmed homogeneity and homoscedasticity.

Preparation of Conidial Suspension of C. coccodes
A conidial suspension of C. coccodes was prepared as per Sánchez-Hernández et al. [109], with minor modifications. Conidia were obtained from 1-week-old PDB cultures (200 mL broth kept in the dark at 25 • C and 140 rpm in an orbital stirrer incubator). The suspension was filtered through two layers of sterile muslin to remove somatic mycelia. Spore concentration was determined using a hemocytometer (Weber Scientific International Ltd.; Teddington, Middlesex, UK), and adjusted to a final concentration of 1 × 10 6 spores (conidia)·mL −1 .

Ex Situ Protection of Tomato Fruits
The efficacy of H. stoechas extract was assessed on artificially infected tomato fruits (cv. "Daniela"), cultivated according to EU organic farming regulations by Huerta El Gurullo (Cuevas del Almanzora, Almería, Spain). All the assayed fruits had a similar size (about 75 mm in diameter) and showed no visible disease symptoms. We slightly modified the protocol proposed by Wang et al. [110]. First, the tomatoes were surface disinfected for 2 min using a 3% NaOCl solution. Then, they were rinsed three times with sterile distilled water and dried on sterile absorbent paper in a laminar flow hood. The fruits were divided into four groups: one group was treated with H. stoechas extract at a concentration equal to the MIC determined in vitro (375 µg·mL −1 ) and another group received twice the MIC concentration (750 µg·mL −1 ), while the remaining two groups served as negative and positive controls (no treatment/no pathogen and pathogen/no treatment, respectively). Under aseptic conditions, each fruit was punctured at three equidistant points in the equatorial region using a truncated needle (3 mm diameter × 5 mm depth). The treated fruits were initially filled with 20 µL of the corresponding treatment (at MIC or MIC×2 concentrations, supplemented with 0.2% Tween ® 20). After one hour, wounds were inoculated with 20 µL of a C. coccodes spore suspension (1 × 10 6 conidia·mL −1 ). Positive controls were solely inoculated with the C. coccodes spore suspension, while negative controls were inoculated with sterile deionized water containing 0.2% Tween ® 20. Each fruit was placed in a separate clean container (corresponding to its treatment) with sterile moistened cotton and incubated at 25 • C for ten days. Lesion diameters were measured twice at right angles to one another on the fruit surfaces, and the percentage of lesion size reduction compared to the positive control (0% reduction) was calculated using the formula: LSR (%) = [(LS c − LS t )/LS c ] × 100, where LS c represents the lesion diameter of the positive control, and LS t represents the lesion diameter of the treated fruits. On day 10, at the end of the experiment, the tomatoes were cut open to analyze the internal lesions.
In these experiments, a contrast fungicide was not used, given that, in the Spanish national legislation on registration of phytosanitary products, there is currently no authorized fungicide for direct use in this plant product (postharvest tomatoes).
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules28155861/s1, Figure S1: ATR-FTIR spectrum of Helichrysum stoechas dried inflorescences; Figure S2: GC-MS chromatogram of Helichrysum stoechas hydromethanolic inflorescence extract; Figure S3: Comparison of MS spectra of 4-ethenyl-1,3benzenediol with that of the chemical species detected at RT = 12.779 min; Table S1: Efficacies reported in the literature for plant extracts against the six phytopathogenic fungal taxa under study.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to their relevance to an ongoing Ph.D. thesis.